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United States Department of Agriculture

Agricultural Research Service

Title: Characterization of Recombinant Alpha-Glucosidase Expressed in Pichia Pastoris

Authors
item Muslin, E - UNIVERSITY OF WISCONSIN
item Kanikula, A - UNIVERSITY OF WISCONSIN
item Clark, S - UNIVERSITY OF WISCONSIN
item HENSON, CYNTHIA

Submitted to: Plant Physiology Supplement
Publication Type: Abstract Only
Publication Acceptance Date: March 5, 1998
Publication Date: N/A

Technical Abstract: Alpha-glucosidase has been shown to significantly contribute to starch degradation both in situ and in vitro. Recently two barley (Hordeum vulgare) alpha-glucosidase cDNAs were isolated, cloned into the pPIC9 vector, and expressed in Pichia pastoris (Tibbot and Skadsen, 1996); only one, however, was found to be enzymatically active (rAGL). The active clone (2600 base pairs) is missing 58 amino acids from the N-terminus of the full length clone (2752 base pairs). These 58 amino acids include a 20 amino acid stretch that is predominantly hydrophobic and may be the secretion signal sequence. Various physiochemical properties of the recombinant enzyme were compared to those of the barley enzyme(s). The Ea of the high and low pI isoforms from barley and the rAGL are 11.8, 6.2 and 7.2 kcal per mol, respectively. Fifty percent of enzyme activity is lost after a 10 min exposure to 38, 45, and 50 C for the barley high and low pI isoforms and the rAGL, respectively. Optimal activity of rAGL is at pH 6 while the pH optima of the barley high and low pI isoforms are 4.1 and 4.3, respectively. Future work will concentrate on the recombinant enzyme and its industrial relevance with the special emphasis on thermostability and substrate specificity.

Last Modified: 8/19/2014
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